NextSeq 550 paired end sequencing; CUT&RUN Negative Controls in Human and Mouse
CUT&RUN Negative Controls in Human and Mouse
HEK293T cells were harvested using TrypLE (Cat. #12604013, Gibco). Tissues were dissociated to single-cell via incubation in Tryple Express Enzyme (12-604-013, Thermo Fisher Scientific; 1 ml /4 limbs) for 15 minutes at 37C °C on a shaker. The cell suspension was resuspended in ice-cold PBS, filtered through a 40 m cell strainer (KKE3.1, Carl Roth) and further processed for CUT&RUN. The age of embryos was determined according to timed mating and vaginal plug observation (E0.5 dpc) and confirmed by morphological criteria. Pregnant females were sacrificed by cervical dislocation and E11.5 embryos were surgically removed. Tissues were dissected under a dissection stereomicroscope (SZ61, Olympus). Samples were washed twice in wash buffer (HEPES pH 7.5 [20 mM], NaCl [150 mM], Spermidine [0.5 mM], Roche Complete Protease Inhibitor EDTA-Free (Cat. # COEDTAFRO, Roche)) and bound to 20 µl Magnetic ConA Agarose beads (Cat. #ABIN6952467, antibodies-online) equilibrated in binding buffer (HEPES pH 7.5 [20 mM], KCl [10 mM], CaCl2 [1 mM], MnCl2 [1 mM]). Antibody incubation was performed in 150 µl antibody buffer (wash buffer with digitonin [0.01%] and EDTA [2 mM]) with 1.5 µl anti-β-catenin antibody (Cat. #ABIN2855042, antibodies-online) overnight at 4 °C. Thereafter washes were performed with wash buffer containing 0.01% digitonin. After overnight incubation, samples were washed three times and pAG-MN protein was added at 0.6 µg/ml, in total volume of 150 µl. pAG/MNase was a gift from Steven Henikoff (Addgene plasmid #123461; http://n2t.net/addgene:123461; RRID: Addgene_123461), expressed and purified as described in (REF Meers 2019a). Samples were washed three times, followed by digestion for 30 min in wet ice in wash buffer supplemented with 2 mM CaCl2. 2X stop buffer (NaCl [340 mM], EDTA [20 mM], EGTA [4 mM], digitonin [0.05%], RNase A [100 µg/ml], glycogen [50 µl/ml]) was added to stop the reaction, and samples were incubated for 30 min at 37 °C. Beads were collected on the magnet and liquid transferred to a new tube. 2.5 µl proteinase K (Cat. #P8107S, New England BioLabs) and 2 µl 10% SDS were added, and samples incubated for 1 hr at 50 °C. Cells were washed three times in Nuclear Extraction (NE) buffer (HEPES-KOH pH-8.2 [20 mM], KCl [10 mM], Spermidine [0.5 mM], IGEPAL [0.05%], Glycerol [20%], Roche Complete Protease Inhibitor EDTA-Free), resuspended in 40 µl NE per sample and bound to 20 µl Magnetic ConA Agarose beads equilibrated in binding buffer. After incubation nuclei and beads are resuspended for 5 min in EDTA wash buffer (wash buffer with EDTA [0.2 mM]). Samples were divided in 200 µl PCR tubes and antibody incubation was performed in 200 µl wash buffer with 2 µl of antibody (for antibody and batch information, see Table 1) ON at 4 °C on a rotator. After ON incubation samples were washed 5 times in wash buffer and resuspended in 200 µl of pAG-MN buffer (wash buffer with pAG-MN 0.6 µg/ml) for 30 min at 4 °C on a rotator. Samples were washed five times, followed by digestion for 30 min in wet ice in wash buffer with 2 mM CaCl2. After 30 min the digestion buffer was removed and the reaction was stopped with 50 µl of 1X Urea STOP buffer (NaCl [100 mM], EDTA [2 mM], EGTA [2 mM], IGEPAL [0.5%], Urea [8.8 M]) and the samples were incubated 1 hr at 4°C. Beads were collected on the magnet and liquid transferred to a new PCR tube in which it was cleaned up twice using Mag-Bind TotalPure NGS beads (Cat. #M1327, Omega Bio-Tek) at 2X, and then resuspended in 20 µl Tris-HCl pH 7.5. Cells were washed three times in Nuclear Extraction (NE) buffer (HEPES-KOH pH-8.2 [20 mM], KCl [10 mM], Spermidine [0.5 mM], IGEPAL [0.05%], Glycerol [20%], Roche Complete Protease Inhibitor EDTA-Free), resuspended in 40 µl NE per sample and bound to 20 µl Magnetic ConA Agarose beads equilibrated in binding buffer. After incubation nuclei and beads are resuspended for 5 min in EDTA wash buffer (wash buffer with EDTA [0.2 mM]). Samples were divided in 200 µl PCR tubes and antibody incubation was performed in 200 µl wash buffer with 2 µl of antibody (for antibody and batch information, see Table 1) ON at 4 °C on a rotator. After ON incubation samples were washed 5 times in wash buffer and resuspended in 200 µl of pAG-MN buffer (wash buffer with pAG-MN 0.6 µg/ml) for 30 min at 4 °C on a rotator. Samples were washed five times, followed by digestion for 30 min in wet ice in wash buffer with 2 mM CaCl2. After 30 min the digestion buffer was removed and the reaction was stopped with 50 µl of 1X Urea STOP buffer (NaCl [100 mM], EDTA [2 mM], EGTA [2 mM], IGEPAL [0.5%], Urea [8.8 M]) and the samples were incubated 1 hr at 4°C. Beads were collected on the magnet and liquid transferred to a new PCR tube in which it was cleaned up twice using Mag-Bind TotalPure NGS beads (Cat. #M1327, Omega Bio-Tek) at 2X, and then resuspended in 20 µl Tris-HCl pH 7.5. Library preparation was performed using the KAPA Hyper Prep Kit for Illumina platforms (Cat. #KK8504, KAPA Biosystems) according to the manufacturer's guidelines with the following modifications. End repair and A-tailing was performed with 0.4X volume reactions with 20 µl of purified DNA. The thermocycler conditions were set to 12 °C for 15 min, 37 °C for 15 min and 58 °C for 25 min to prevent thermal degradation of the shortest fragments. Adapter ligation was performed in 0.4X volume reactions. KAPA Dual Indexed adapters were used at 0.15 µM. A post-ligation clean-up was performed with Mag-Bind TotalPure NGS beads at 1.2X the sample volume. Resuspension was done in 10 mM Tris-HCl pH 8.0. Library amplification was performed in 0.5X volume reactions. The cycling conditions were set as follows: 45 sec initial denaturation at 98 °C, 15 sec denaturation at 98 °C, 10 sec annealing/elongation at 60 °C, 1 min final extension at 72 °C, hold at 4 °C, with 13 cycles. After amplification, a post-amplification cleanup was performed using NGS beads at 1.2X sample volume. Libraries were then run on an E-Gel EX 2% agarose gel (Cat. # G402022, Invitrogen) for 10 min using the E-Gel Power Snap Electrophoresis System (Invitrogen). Bands of interest between 150 and 500 bp were excised and purified using the GeneJET Gel Extraction Kit (Cat. #K0691, Thermo Scientific) according to manufacturer's instructions.